Neonatal encephalopathy (NE) is a serious condition with life long consequences. NE is the most important cause of morbidity and mortality in the term born baby. About 10% of those affected die and 25% are severely handicapped due to long-term complications such as cerebral palsy, mental retardation with learning difficulties, cerebral visual impairments and/or epilepsy. Its long-term consequences impose a large burden on the child and family and on worldwide healthcare budgets. The estimated costs of the treatment of sequelae of NE in the US is around 750 000 US $ per patient. One major reason for NE is pre-, peri- and postnatal asphyxia, a condition in which the fetus or newborn lacks oxygen. In the Western world about 0.9% of newborns, about 130.000 in the developed world and 30.000 in European Union suffer from a moderate to severe form of perinatal asphyxia. However NE could not always be explained by moderate to severe asphyxia and the cause of NE often could not be identified. It has been shown that foetuses/newborns with rather mild asphyxia, who initially seem to recover without complications, will have behavioural problems in childhood, which can be traced back to the perinatal insult.
A study published in Lancet suggests that the definition of NE does not include all patients with long-term neurological outcome after resuscitation (Odd D E, Lewis G, Whitelaw A, Gunnell D. Lancet. 2009 May 9; 373(9675):1615-22). Resuscitated infants asymptomatic for the “classically defined” encephalopathy, however, undergoing actual brain damage (or prone to actual brain damage) might result in a larger proportion of adults with low IQs than do those who develop neurological symptoms consistent with encephalopathy. Based on this publication 40% of cases with long-term adverse neurological outcome are missed by current, state of the art, diagnostic means. Thus, in summary neonatal encephalopathy and its long-lasting consequences is a huge burden to the child, to its family. Consequently, there is a lack of early markers identifying newborns at risk for NE.
Till date, therapy was limited to supportive care including adequate oxygenation and restoring of circulation by appropriate, rapid and effective resuscitation. It is vital to maintain adequate ventilation, systemic blood pressure, tissue perfusion, and normoglycaemia, to control seizures, and to correct electrolyte and acid-base disorders. However, in the past few years hypothermia (whole body hypothermia or selective head cooling), has been introduced to reduce morbidity and mortality following perinatal asphyxia in term born neonates. In a recent meta-analysis combining the results of 1320 neonates the effect of moderate hypothermia was found to be associated with a moderate reduction in death and neurological impairment at 18 months. It is of outmost importance that the therapeutic benefit of hypothermia is strongly dependent on the time point of its initiation. The earlier the diagnosis is made and the early therapy can be started the more neuroprotection can be achieved. Accordingly, early detection of neonatal encephalopathy and severity assessment is vital to therapeutic success.
Current diagnostic tests to evaluate whether a newborn is at risk to develop neurological sequelae have major limitations with low sensitivity and specificity. The diagnostic criteria for neonatal encephalopathy in term newborn infants require i) signs of perinatal and postnatal asphyxia, abnormal blood gas values (increase in base deficit, blood lactate values, low cord pH in the umbilical artery (UApH) and need for resuscitation, and ii) signs of brain involvement and abnormal neurology characterized by neurological scores (Sarnat or Thompson score) and any presence of seizure activity and iii) demonstrating electroencephalographic evidence of abnormal cerebral function by means of the amplitude-integrated or ten-lead standard EEG. These established tests, however, have major limitations. We have recently shown that that so called “abnormal blood gas values” do not correlate with the extent of hypoxia. This is reflected by low sensitivity and specificity for NE [Groenendaal, de Vroes Semin Neonatol 2000, Vol 5 17-32]. The most precise test currently established is the use of amplitude integrated EEG (aEEG) with a sensitivity and specificity of 80% in the first 6 hours of life. The use of aEEG is challenged by its availability, required expertise and timing. It is not available in all children's hospitals and in none of the delivery units. Thus the diagnosis by aEEG can only be made after transfer into a children's clinic in which the tool and the medical expertise is available. In addition the process of implementation of the aEEG and the evaluation of the signal takes at least 30 to 45 minutes. This is of particular importance since it is known that treatment is more effective the earlier it is initiated. Imaging technologies such as MRT (Magnetic Resonance Tomography), however, are applicable from day three after birth only and therefore, are not useful for early diagnosis and timely therapy.
Currently used diagnostic methods thus require time and appropriate equipment with high costs, do not recognize affected parts of the brain along with individual prognosis, afford results too late for appropriate therapy and with frequently unsatisfying sensitivities. These available diagnostic means, therefore, have major limitations such as reduced area under the curve (AUC) and/or delay of diagnosis or increased costs due to equipment required. Accordingly, these procedures also do not allow a timely assessment of an acute and rapidly evolving disease nor a differentiation of brain areas affected by hypoxia. Overall the situation is far from satisfying and from providing a rapid, reliable and precise diagnosis of brain damage in neonates, let alone a differentiation of affected brain areas or tissues, a prerequisite, however, for selection and initiation of appropriate therapy; there is still an urgent need for differentiation of brain injury from any other state of health to enable timely and adequate treatment.
Due to these limitations academic research groups have been looking for potential alternative biomarkers of NE like interleukin-1b, 6, 8, 9, 12, NSE, S100, CK-BB, Phosphorylated axonal neurofilament heavy chain (pNF-H protein2) Ubiquitin C-terminal hydrolase 1 (UCHL1 protein) (Ramaswamy et al. (2009) Pediatr Neurol, Vol 40, 215-226). However, although these markers have been published, none of these were finally developed to a diagnostic product because of major limitations. The studies are not uniform and show a significant amount of heterogeneity with a tremendous variation in the assessment of outcomes and variation in inclusion criteria. Most frequently the inclusion of patients was based on “established” tests. Thus most of the studies confirm the diagnosis of the established tests and were most precise for infants with severe injury who are already easily identified and do not provide any additional information. Most of the studies had small sample size, so it is not possible to calculate true predictive value for all of the mentioned biomarkers and no attempt has been made to correlate concentrations of these marker candidates with brain areas affected by NE. In addition, correlation is strongest at times well after the latent phase, which is way too late for early and therefore, effective treatment.
In summary there is no single precise biomarker available to diagnose or predict neonatal encephalopathy in newborns at an early stage e.g. immediately after birth or any biomarker which provides information on brain areas affected by damaging conditions. Lesions detectable on a biochemical level only such as by using endogenous metabolites as biomarkers and not monitorable by alternate means, however, can be used to assess neurological outcome which is of outstanding value for clinical diagnosis.
In classical patient screening and diagnosis, the medical practitioner uses a number of diagnostic tools for diagnosing a patient suffering from a certain disease. Among these tools, measurement of a series of single routine parameters, e.g. in a blood sample, is a common diagnostic laboratory approach. These single parameters comprise for example enzyme activities and enzyme concentration and/or enzyme detection
As far as such diseases are concerned, which easily and unambiguously can be correlated with one single parameter or a few number of parameters achieved by clinical chemistry, these parameters have proved to be indispensable tools in modern laboratory medicine and diagnosis. However, in complex pathophysiological conditions, for which an unambiguously assignable single parameter or marker is not available, differential diagnosis from blood or tissue samples is currently difficult to impossible.
Only recently metabolomic analyses for specific diagnostic approaches were described in the prior art:
According to WO 2011/012553 A1 a method for predicting the likelihood of an onset of an inflammation associated organ failure is provided, which is based on quantitative metabolomics analysis of a biological sample of a mammalian subject in vitro. In particular, the concentration of acylcarnitines, sphingomyelins, hexoses and glycerophospholipids in plasma by means of FIA-MS/MS is determined. Furthermore, amino acids and biogenic amines were analyzed by reversed phase LC-MS/MS in plasma. Prostanoids—a term summarizing prostaglandins (PG), thromboxanes (TX) and prostacylines—and oxidised fatty acid metabolites in plasma extracts were analysed by LC-ESI-MS/MS and in brain homogenate extracts by online solid phase extraction (SPE)-LC-MS/MS. Furthermore, energy metabolism (organic acids) was analyzed by LC-MS/MS. For the quantitative analysis of energy metabolism intermediates (glycolysis, citrate cycle, pentose phosphate pathway, urea cycle) hydrophilic interaction liquid chromatography (HILIC)-ESI-MS/MS method was applied.
WO 2010/128054 A1, corresponding to EP 2 249 161 A1, describes a method of diagnosing asphyxia. In particular, said document refers to a method for in vitro diagnosing e.g. perinatal asphyxia and disorders related to hypoxia, characterized by quantitatively detecting in at least one biological sample of at least one tissue of a mammalian subject a plurality of asphyxia specific compounds having a molecular weight of less than 1500 Dalton, except lactate, comprising the steps of:
a) selecting said compounds;
b) measuring at least one of the parameters selected from the group consisting of: concentration, level or amount of each individual metabolite of said plurality of metabolites in said sample, qualitative and/or quantitative molecular pattern and/or molecular signature; and using and storing the obtained set of values in a database;c) calibrating said values by comparing asphyxia-positive and/or asphyxia-negative reference parameters;d) comparing said measured values in the sample with the calibrated values, in order to assess whether the patient is asphyxia-positive or asphyxia-negative.
The method according to WO 2010/128054 A1 uses asphyxia specific compounds as biomarkers which are endogenous compounds being selected from the group consisting of: biogenic amines; carnitine-derived compounds; amino acids; bile acids; carboxylic acids; eicosanoids; lipids; precursors of cholesterol, cholesterol metabolites, prostanoids; and sugars. Furthermore, WO 2010/128054 A1 relates to a method of in vitro estimating duration of hypoxia in a patient, a method for in vitro monitoring of normoxic, hypoxic and hyperoxic conditions and/or normobaric and hyperbaric oxygen therapy and a kit for carrying out the methods thereof.
However, neither assignment of metabolite concentrations to total brain damage or NE nor to distinct brain regions nor neurological outcome in neonates is addressed with the metabolomic studies disclosed in WO 2011/012553 A1 and WO 2010/128054 A1.
Solberg R and colleagues (“Metabolomic Analyses of Plasma Reveals New Insights into Asphyxia and Resuscitation in Pigs”, PLoS ONE, 2010, 5(3)) disclose detection of a number of metabolites in plasma taken before and after hypoxia as well as after resuscitation, in asphyxiated piglets, in order to evaluate pathophysiological mechanisms of hypoxemia in newborns. Hypoxemia of different durations was induced in newborn piglets before randomization for resuscitation with 21% or 100% oxygen for 15 min in order to detect markers of the duration/severity of hypoxia and to detect markers of therapy response due to different resuscitation protocols. The metabolites of the study of Solberg et al. includes amino acids, particularly branched chained amino acids, metabolites of the Krebs cycle, including alpha-ketoglutarate, succinate and fumarate, biogenic amines, bile acids, prostaglandins, sphingolipids, glycerophospholipids, oxysterols and acylcarnitines. Assessment of brain injuries per se, or biomarkers to detect brain injuries per se are not comprised by the Solberg et al. paper (cf. page 9, left column, lines 2-3), and identification or differentiation of brain areas or tissues or assessment of neurological outcome is not addressed.
Beckstrom et al. (J ChromatogrA, 2011 Vol 1218, 1899-1906) evaluated whether metabolomic profiling can reveal metabolite changes in plasma after asphyxia in a Macaca nemestrina model of perinatal asphyxia. The metabolic profile of post-asphyxia samples showed marked variability compared to the pre-asphyxia samples. This metabolomic analysis confirmed lactate and creatinine as markers of asphyxia and discovered new metabolites including succinic acid and malate (intermediates in the Krebs cycle) and arachidonic acid (a brain fatty acid and inflammatory marker). Although these metabolite changes reflect the changes of asphyxia (similarly to the publication of Solberg et. al.), the metabolite changes were not related to brain injury, to identification or differentiation of brain areas or tissues or assessment of neurological outcome.
Chu, C. Y et al. (Clinical Biochemistry, 2006, Vol 39, 203-209) describe metabolomic and bioinformatic analyses in asphyxiated neonates. In particular they analyzed urine of such neonates and defined eight urinary organic acids which were significantly associated with the prognosis of neurodevelopmental handicap with high sensitivity and specificity. They further divided said acids into two classes of acids, one consisting of acids which were associated with good neonatal outcome (ethylmalonate, 3-hydroxy-3-methylglutarate, 2-hydroxyglutarate, 2-oxogluturate) and the other with poor outcome (glutarate, methylmalonate, 3-hydroxybutyrate, orotate). No blood samples were analyzed; nor were said urinary metabolites or combinations thereof correlated to brain injury, injured areas of the brain or any neurological behaviour scoring. However all of these metabolites we determined only in urine samples, and, moreover, not within the first hours of life.
Finally, Mueller et. al. (“Mass Spectrometric Quantifications of Organic Acids and Acylcarnitines in Early Random Urine Specimens of Newborns with Perinatal Complications: Feasability Study for the Prediction of the Neuro-Developmental Outcome”, The Internet Journal of Pediatrics and Neonatology, 20077(2)) describe the use of mass spectrometric quantifications of organic acids and acylcarnitines for the prediction of the neuro-developmental outcome in newborns with perinatal complications. This group investigated a number of 65 quantitatively determined metabolites (42 organic acids, 22 acylcarnitines, free carnitine and 15 ratios) in urine of infants within the first 72 hours of life of infants. Reliable prediction for development of NE caused by severe asphyxia was demonstrated with metabolite monitoring of the lactic acid/creatinine ratio in urine of asphyxiated newborns. However, an unexpected result of the Mueller et al. study was the finding that the total amount of urinary acylcarnitines did not significantly differ between the comparison group and the patient group with severe neurological defects. Blood metabolite concentration changes related to brain injury, to identification or differentiation of brain areas or tissues, or assessment of neurological outcome were not described. Müller et al. observed a limited number of metabolic carboxylic acid combinations for predicting the neurological outcome of preterm and term newborns at the end of the first year of life. Said metabolite combinations comprise lactic acid in combination with one or more of 3-hydroxybutyric acid, 3-hydroxyisovaleric acid, methylmalonic acid, 4-hydroxyphenyllactic acid, and 5-oxoproline. Again, all of these metabolites we determined only in urine samples, and, moreover, not within the first hours of life.
In summary so far no metabolic markers or marker signatures have been identified for indicating and diagnosing NE at a time point as early as possible (defined as within the first 6 hours after the insult) in infants. No teaching is found in the prior art suggesting that there might exist markers applicable to the identification of neonatal encephalopathy and/or differentiation of brain tissues altered by hypoxic conditions. Solely a couple of intermediates, possibly involved in pathobiochemistry, have been discussed in the wider context of brain damage and chemical mediators that may contribute to gray matter injury.
Thus, the problem underlying the present invention is to provide an early diagnostic approach for assessing NE in infants with high sensitivities and specificities, capable of determining brain tissues involved and/or brain tissues damaged and/or prone to subsequent future damage and/or predict neurological outcome.